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Nucleocytoplasmic recycling of the nuclear localization signal receptor alpha subunit in vivo is dependent on a nuclear export signal, energy, and RCC1.

Boche I, Fanning E - J. Cell Biol. (1997)

Bottom Line: Recombinant Rch1 microinjected into Vero or tsBN2 cells was found primarily in the cytoplasm.After nuclear injection, the truncated Rch1 was retained in the nucleus, but either Rch1 residues 207-217 or a heterologous nuclear export signal, but not a mutant form of residues 207-217, restored nuclear export.However, free Rch1 injected into nuclei of tsBN2 cells at the nonpermissive temperature was exported.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, USA.

ABSTRACT
Nuclear protein import requires a nuclear localization signal (NLS) receptor and at least three other cytoplasmic factors. The alpha subunit of the NLS receptor, Rag cohort 1 (Rch1), enters the nucleus, probably in a complex with the beta subunit of the receptor, as well as other import factors and the import substrate. To learn more about which factors and/or events end the import reaction and how the import factors return to the cytoplasm, we have studied nucleocytoplasmic shuttling of Rch1 in vivo. Recombinant Rch1 microinjected into Vero or tsBN2 cells was found primarily in the cytoplasm. Rch1 injected into the nucleus was rapidly exported in a temperature-dependent manner. In contrast, a mutant of Rch1 lacking the first 243 residues accumulated in the nuclei of Vero cells after cytoplasmic injection. After nuclear injection, the truncated Rch1 was retained in the nucleus, but either Rch1 residues 207-217 or a heterologous nuclear export signal, but not a mutant form of residues 207-217, restored nuclear export. Loss of the nuclear transport factor RCC1 (regulator of chromosome condensation) at the nonpermissive temperature in the thermosensitive mutant cell line tsBN2 caused nuclear accumulation of wild-type Rch1 injected into the cytoplasm. However, free Rch1 injected into nuclei of tsBN2 cells at the nonpermissive temperature was exported. These results suggested that RCC1 acts at an earlier step in Rch1 recycling, possibly the disassembly of an import complex that contains Rch1 and the import substrate. Consistent with this possibility, incubation of purified RanGTP and RCC1 with NLS receptor and import substrate prevented assembly of receptor/substrate complexes or stimulated their disassembly.

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(A) Export of Rch1 is temperature dependent. Rch133–529 and Rch1244–529 were injected into the nuclei of Vero cells at 0°C. The  cells were either fixed (a and c) or incubated on ice for 30 min and then fixed (b and d). Cells were immunostained and fluorescent micrographs were taken. (B) Export of Rch1 is impaired at 0°C. The graph shows the quantification of the nuclear fluorescence of cells injected into the nucleus with Rch133–529 or Rch1244–529 and incubated on ice as indicated on the x-axis before fixing and immunostaining.
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Figure 6: (A) Export of Rch1 is temperature dependent. Rch133–529 and Rch1244–529 were injected into the nuclei of Vero cells at 0°C. The cells were either fixed (a and c) or incubated on ice for 30 min and then fixed (b and d). Cells were immunostained and fluorescent micrographs were taken. (B) Export of Rch1 is impaired at 0°C. The graph shows the quantification of the nuclear fluorescence of cells injected into the nucleus with Rch133–529 or Rch1244–529 and incubated on ice as indicated on the x-axis before fixing and immunostaining.

Mentions: If export of Rch1 from the nucleus depends on the nuclear export signal that is deleted in Rch1244–529, one might also expect the export process, like the nuclear import process, to be temperature dependent. To address this question, we tested export of the NLS receptor α subunit at 0°C. Vero cells were shifted to 0°C immediately before nuclear microinjection of Rch133–529, and Rch1244-529 and then incubated on ice for various time periods. Cells were fixed and the exogenous tagged Rch1 was visualized by immunofluorescence. Cells injected with both forms of Rch1 showed nuclear staining not only immediately after injection (Fig. 6 A, a and c), but also after a 30-min incubation on ice (Fig. 6 A, b and d). Quantitative evaluation of nuclear fluorescence in cells injected with Rch133–529 and Rch1244–529 confirmed that the intensity of nuclear fluorescence was constant over the 30-min time period (Fig. 6 B). These results support the idea that recycling of Rch1 is temperature dependent and show that neither Rch133–529 nor Rch1244–529 can exit the nucleus by simple diffusion.


Nucleocytoplasmic recycling of the nuclear localization signal receptor alpha subunit in vivo is dependent on a nuclear export signal, energy, and RCC1.

Boche I, Fanning E - J. Cell Biol. (1997)

(A) Export of Rch1 is temperature dependent. Rch133–529 and Rch1244–529 were injected into the nuclei of Vero cells at 0°C. The  cells were either fixed (a and c) or incubated on ice for 30 min and then fixed (b and d). Cells were immunostained and fluorescent micrographs were taken. (B) Export of Rch1 is impaired at 0°C. The graph shows the quantification of the nuclear fluorescence of cells injected into the nucleus with Rch133–529 or Rch1244–529 and incubated on ice as indicated on the x-axis before fixing and immunostaining.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139786&req=5

Figure 6: (A) Export of Rch1 is temperature dependent. Rch133–529 and Rch1244–529 were injected into the nuclei of Vero cells at 0°C. The cells were either fixed (a and c) or incubated on ice for 30 min and then fixed (b and d). Cells were immunostained and fluorescent micrographs were taken. (B) Export of Rch1 is impaired at 0°C. The graph shows the quantification of the nuclear fluorescence of cells injected into the nucleus with Rch133–529 or Rch1244–529 and incubated on ice as indicated on the x-axis before fixing and immunostaining.
Mentions: If export of Rch1 from the nucleus depends on the nuclear export signal that is deleted in Rch1244–529, one might also expect the export process, like the nuclear import process, to be temperature dependent. To address this question, we tested export of the NLS receptor α subunit at 0°C. Vero cells were shifted to 0°C immediately before nuclear microinjection of Rch133–529, and Rch1244-529 and then incubated on ice for various time periods. Cells were fixed and the exogenous tagged Rch1 was visualized by immunofluorescence. Cells injected with both forms of Rch1 showed nuclear staining not only immediately after injection (Fig. 6 A, a and c), but also after a 30-min incubation on ice (Fig. 6 A, b and d). Quantitative evaluation of nuclear fluorescence in cells injected with Rch133–529 and Rch1244–529 confirmed that the intensity of nuclear fluorescence was constant over the 30-min time period (Fig. 6 B). These results support the idea that recycling of Rch1 is temperature dependent and show that neither Rch133–529 nor Rch1244–529 can exit the nucleus by simple diffusion.

Bottom Line: Recombinant Rch1 microinjected into Vero or tsBN2 cells was found primarily in the cytoplasm.After nuclear injection, the truncated Rch1 was retained in the nucleus, but either Rch1 residues 207-217 or a heterologous nuclear export signal, but not a mutant form of residues 207-217, restored nuclear export.However, free Rch1 injected into nuclei of tsBN2 cells at the nonpermissive temperature was exported.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, USA.

ABSTRACT
Nuclear protein import requires a nuclear localization signal (NLS) receptor and at least three other cytoplasmic factors. The alpha subunit of the NLS receptor, Rag cohort 1 (Rch1), enters the nucleus, probably in a complex with the beta subunit of the receptor, as well as other import factors and the import substrate. To learn more about which factors and/or events end the import reaction and how the import factors return to the cytoplasm, we have studied nucleocytoplasmic shuttling of Rch1 in vivo. Recombinant Rch1 microinjected into Vero or tsBN2 cells was found primarily in the cytoplasm. Rch1 injected into the nucleus was rapidly exported in a temperature-dependent manner. In contrast, a mutant of Rch1 lacking the first 243 residues accumulated in the nuclei of Vero cells after cytoplasmic injection. After nuclear injection, the truncated Rch1 was retained in the nucleus, but either Rch1 residues 207-217 or a heterologous nuclear export signal, but not a mutant form of residues 207-217, restored nuclear export. Loss of the nuclear transport factor RCC1 (regulator of chromosome condensation) at the nonpermissive temperature in the thermosensitive mutant cell line tsBN2 caused nuclear accumulation of wild-type Rch1 injected into the cytoplasm. However, free Rch1 injected into nuclei of tsBN2 cells at the nonpermissive temperature was exported. These results suggested that RCC1 acts at an earlier step in Rch1 recycling, possibly the disassembly of an import complex that contains Rch1 and the import substrate. Consistent with this possibility, incubation of purified RanGTP and RCC1 with NLS receptor and import substrate prevented assembly of receptor/substrate complexes or stimulated their disassembly.

Show MeSH
Related in: MedlinePlus